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Team:INSA Toulouse/contenu/lab practice/notebook/protocols/charac recomb
From 2013.igem.org
Notebook
Protocols
In vitro recombinase characterization protocol
Recombinase overexpression and extraction
- The E.coli K12-DH5.1 containing recombinase and Cloramphenicol resistance genes were grown in variable amount of Chloramphenicol LB culture medium.
- After 4h growth at 37°C, the culture was centrifuged for 3 min at 13000 rpm.
- The cell pellets were resuspended in 500µl Rec buffer containing 20 mM Tris, pH 7.5, 10mM EDT1, 25mM NaCl, 10mM Spermidine, 1mM DTT and 0.1g.mol-1 BSA.
- The cells were lysed by sonication for three bursts of 30s.
- After centrifugation for 15 min at 13000 rpm, 4°C, the supernatant containing proteins (recombinase) was collected and incubated on ice.
In vitro recombination test
- Recombination reactions were assembled on ice with recombinase protein extraction solution and a variable amount of solution containing Rec buffer and the gate plasmid.
- Reactions were incubated at 37°C for up to 2 hours and heat inactivated at 75°C for 10min.
- Reactions were transformed into E.coli K12-DH5.1.
- Spread out the cells on Kanamycine LB agar plate.
- The plate was incubated overnight at 37°C.
Note: For Xor1 gate characterization, the in vitro recombination test was performed at different Bxb1 concentrations (100µl and 20µl of Bxb1 extraction solution) and different hours of reactions (2 hours and 5 hours).
* To know which antibiotics to use, look at the plasmid that the part is in. The naming scheme for plasmids is specifically designed to indicate antibiotic resistance.
Note: There is not enough DNA in each well to perform anything but transformations.
